Spectrophotometry process



Nov. 17, 1959 R. H. HAMILTON SPECTROPHOTOMETRY PROCESS Filed March 18,1954 United States Patent This invention relates to improvements inspectrophotometry, and a principal object of the invention is to providean improved process of displacement or absorption spectrophotometry ashereinafter set forth.

One of the problems encountered in laboratories using test tube typeabsorption cells for spectrophotometry is the securing and maintenanceof sets of matched tubes of sufiiciently close characteristics. Tocircumvent this difficulty, displacement plates can be usedtoallow'displacement and replacement of layers of the colored fluid ofuniform thickness. Critical study of the use of such plates shows a highdegree of accuracy to be attainable. Results further indicate thepossibility of eliminating need for transferring photometric solutionsfrom the "Working test tubes in which previous analytical steps werecarried out team optical set of test tubes in which photometric readingsare made. Limitations of the method are discussed, and possibilities areconsidered for selection of suitable displacement plates.

The invention will be more readily understood by reference to theattached drawings, wherein: Fig. 1 is a sectional elevational viewshowing apparatus capable of use in the practice of my invention, andillustrating an initial step of the operation; Fig. 2 is a correspondingview illustrating a subsequent step; and Fig. 3 is a transversesectional view on the line 3-3, Fig. 1.

In the usual absorption photometry of solutions, light of restrictedwave length incident on the photocell or phototube is set to read unitintensity after passing through a cell containing solvent or solventplus the amount of impurities in reagents (blank). Then, light intensitybeing maintained constant, an identical cell is substituted, containingsolvent plus the light-absorbing molecules whose concentration is to bedetermined. The decrease in light intensity is noted and theconcentration of the solute is calculated from the absorption producedby knownconcentrations.

The same results can be obtained by the addition to the light path oflayers of solution of constant thickness. Such addition can beaccomplished by removal of adisplacement plate immersed in the solution.By way of example, reference may be had to the drawings, Figure -1 ofwhich shows an ordinary test tube 1, somewhat wider than the beam 3 ofmonochromatic light, or light of restricted wavelength presented by aspectrophotometer; The tube, containing a solution 4 the optical densityof which it is desired to determine, is centered in a stable position inthe light beam of the spectrophotometer. A displacement plate 2 shown inlongitudinal section through the narrower dimension, and being arectangular strip of plate glass or similar substance as discussedherein, narrow enough to fit into the tube, and wider than the beam ofmonochromatic light, is set into the solution in such a way as to beperpendicular to the light beam. The length of light path through theabsorbing solution is obviously A plus B. The distance L, being thethickness of the displacement plate, and the plate beingmade of amaterial of the displacement plate.

. 2,912,895 Patented Nov. 17, 1959 transparent to light of thewave-length used, does not contribute appreciably to the absorption ofthe light used. Provision is made in the use of the displacement plateto compensate for this small absorption of light in the distance LyFigure 2 shows everything shown in Figure 1, in the .same position andunder the same circumstances, except only that the displacement platehas been withdrawn. The length of light path thro'ughthe absorbingsolution is now A plus B plus L. The change in length of the light paththrough the absorbing solution is obviouslyL. This distance isindependent'of A, of B, and of A plus B. In elfect, by removal of thedisplacementplate, one has interposed into the light path a rectangularlayer of absorbing solution of the samedimensions as to width andthickness as the removed displacement plate. Within the limitationsdiscussed elsewhere, the effect of this interposed layer of solutionwillbe independent of the size of, and of irregularities in, the testtube 1. It thus becomes unnecessary to use expensive rectangular opticalcells, or carefully selected tubes, to contain the absorbing solution,since one can by using a displacement plateas described always interposein the light beam a constant, reproducible layer of absorbing solution.0

Figure 3 shows a transverse sectional view at the level of the lightbeam (3-3 in Figure 1), with the displace ment plate in position.

For a given solution and wave length the effect of the displacementplate itself onlight transmittance will be constant. Either of twoprocedures can be followed: (1) With the displacement plate immersed atright angles to the light beam, light intensity is set to read 100%(unity). The plate is then removed, and light intensity is read afterremoval. (2) Light intensity is allowed to remain such thattransmittance is close to unity (between and and transmittance is readexactly, before and again after removal of the displacement plate. Thedifierence inthe optical densities correspondingto .the twotransmittances, corrected by a similar density difierence obtained witha blank, gives an optical density corresponding tothat of the layer ofsolution displaced by the plate.

It is possible to use the latter procedure because of the followingmathematical relationship:

and D =log T /T =log T +log T The optical density change produced byremoving the plate is Hence, even though T is much larger than unity(100%) if T and T fall on the scale, it is still possible, from thedifference in their logarithms, to obtain the optical density value ofthe displaced solution.

Provided'the cell is not moved during removal of the displacement plate,and provided the latter is positioned in a plane perpendicular to thelight beam and is larger than the light beam, the accuracy of resultsobtained should be dependent only on the precision of determining thechange in light density produced by removal Hence even scratched testtubes can be used. Furthermore, because the change in density isdetermined only by the characteristics of the for exact uniformity ofsize.

Ideally the plate should be in a plane perpendicular, or normal to thelight beam. Rotation of the plate from the plane will produce (1)variation in the reflectance at entrance and exitsurfaces, and (2)variation in the length of solution displaced due to the longer slantingpath of the light beam through the plate. Factors conerned in these twosources of error are (l) refractive index of the liquid, n (2)refractive index of the displacement plate, 12 and (3) angle ofincidence, 1'.

Take n=n /n Also take E= /n sin i and reflected light incident light;

Fresnels formula can be modified to give R as a function of n and of theangle of incidence:

R: E-cos i) E I-sin 'i tan E+cosi (E-l-sin 2' tan 7!) Assuming for n thevalue 1.33, and for n the value 1.55, n becomes 1.165, and the aboveformula gives values for fraction of light reflected at the entrancesurface, at varying values of i, as shown in Table I under R.

Table I.Vdriatin in light transmittance with angle of incidence, due toreflection R, and length of light path, L

Angle of Incidence, R L

As will be seen, reflection does not change appreciably for smalldeviations in the position of the displacement plate.

The situation is different, however, with respect to the change inlength of the path of the refracted ray through the displacement platewith rotation.

Taking D as the optical density of a layer of the displaced liquid equalin thickness to the displacement plate set normal or perpendicular tothe light ray, and taking D, as the apparent or false optical density ofa thicker layer of liquid corresponding to the longer path of therefracted ray through the rotated plate, we can define the resultingpositive error, L, as

of refraction of the light ray corresponding to the angle of incidence,i, that v Displacement spectrophotometry offers the opportunity formaking accurate relative optical density measurements in unselected testtubes, which can be calibrated for certain volumes, used in the waterbath, employed fo dilution to volume without transfer, and then placeddirectly in the photometer. Displacement plates have been employedpreviously in optical cells but only for the purpose of decreasingthickness of the liquid layer,

.for use with liquids of high optical density. Their use in ordinarytest tubes as described here may make desirable readjustment of theconcentrations of light-ab sorbing molecules to secure an optimumoptical density range. This change can be produced by using a largeraliquot of the sample, by lesser dilution of the final colored solution,or in some cases by using a difierent wave length of light at which theabsorption is greater.

The maximum thickness of displacement plate that can profitably be usedwill be determined by several considerations. The plate should be enoughwider than the light beam used that stray light will not escape aroundthe edges. Furthermore, the width of the plate should be enough to avoidreflection from the edges. Within the limits of the circular crosssection of the tube, the thicker the plate is, the narrower it will haveto be.

Because the plate displaces solution, care will have to be taken thatliquid is not caused to run over when it is inserted. If dilution to amark is made, part of the contents can be spilled into the sink aftermixing, to reach a safe level. Thickness of the plate will determine theamount of displacement, and hence the amount of liquid that can safelybe left in the tube.

The errors in displacement spectrophotometry include in general allthose of other types, with one or two exceptions. An exception is thecontribution to error made by surface dirt or scratches on theabsorption cell surface. These do not, within limits, affect theaccuracy of displacement spectrophotometry.

Errors peculiar to displacement spectrophotometry include dirt,scratches, or bubbles on the displacement plate. These imperfections maycause a loss of uniformity if several plates are used. When a singleplate is used surface imperfections that are constant in effect, such asscratches, should produce little error, whereas changing imperfectionssuch as bubbles will cause errors even when only a single plate is used.

It can be shown that it is not necessary to achieve precision in settingthe plate at right angles to the light beam, but that good accuracy canbe attained by approximating the proper position. Sufficient accuracycan be attained by inspection, without elaborate precautions. Out oftwenty consecutive times in which the plate was set in place byinspection and in which the angle of deviation of the plate fromnormalcy was then measured, in no instance did the deviation exceed 2.At such values of i, the error from this source is negligible.

A more serious source of error is the presence of inequalities in thedisplacement plates used. Ordinary polished plate glass can vary morethan 2% in thickness over very short distances. Glass plates and evenpolished narrow-band-pass filters may deviate 1% or more in theparallelism of the two surfaces. Such lack of parallelism will affectthe integrated thickness of the plate within the light beam. The limitsof this variability in commercial plate glass differ with the method ofmanufacture, and should be leastwith drawn glass finished with largerpolishing blocks. There is a possibility of error arising from localvariations in the index of refraction of glass not prepared for opticalpurposes. These errors can be minimized by using a single displacementplate, which willhave to be wiped dry between tubes containing solutionswith appreciable difierences in concentration. Cleaning would befacilitated by treatment of the surface with a silicone, or by makingthe plate from a transparent nonwctting plastic of suflicient resistanceto chemicals. Care would have to be taken that air bubbles did no clingto such a surface on immersion.

It should not, however, be difiicult to prepare plates of glass,plastic, quartz, or other transparent material, of sufficiently uniformthickness and optical characteristics to be interchangeable. v

'Light absorption of the unused or undisplaced solution will act as anadditional light filter, and may accentuate stray light effects,especially with wide band width. Thus, marked changes in the geometry ofthe containing vessel may affect the apparent density values obtained.

Density difierences obtained as described are not proestates portionalto concentration of solute. Plotting of density difference valuesagainst concentration gives a straight line which in general does notgothrough the origin. The same statement applies to the usual type ofspectrophotometry, unless a blank is used, because of the constantcontribution to each value by the blank absorption. It is customary tocorrect for this blank value, either automatically in the setting of theinitial light intensity (setting the instrument for T=l00% with ablank), or by subtracting the density value of the blank from each ofthe other values. In displacement spectrophotometry this constant valueis the resultant of the reagent blank plus the contribution of the plateitself (absorption by the glass, reflectance, and other factors). It caneasily be determined by measuring the density difference produced byimmersion of the plate in a reagent blank such as is usually prepared.

Ordinary, unselected test tubes can be used for quantitative absorptionspectrophotometry by making density readings before and after removal ofa flat plate of glass or other transparent material set in the tube todisplace a constant depth of solution. Studies of the nature andmagnitude of errors that may be encountered in the procedure show thatwith moderate precautions good accuracy can be anticipated.

I claim:

In the process of spectrophotometric determination of the concentrationof molecules that absorb radiant energy in a fluid of concentrationunknown as regards such molecules by the measurement of absorption ofradiant energy from a narrow beam of radiant energy of suitablyrestricted wavelength passed transversely through a substantiallytransparent container of the fluid, the steps comprising: displacingfluid within said container by inserting a displacement plate withsmooth, parallel, plane surfaces, made of a material absorbing little ofthe radiant energy employed, at right angles across the radiant energybeam to produce within the fluid an optically substantly void space,with smooth, parallel, plane surfaces, transverse to the beam of radiantenergy employed and wholly containing the cross section of said beam;measuring the radiant energy transmitted in said beam through saidcontainer, displacement plate, and undisplaced fluid; withdrawing thedisplacement plate, whereby the unknown fluid replaces the spaceoccupied by the plate, thus producing an increase in the length of pathof the said beam of radiant energy through said fluid correspondingexactly to the distance the beam previously traversed the opticallysubstantially void plate, and thus causing a greater absorption of theradiant energy used; measuring the radiant energy transmitted in saidbeam through said container and fluid with said displacement plateremoved; the change in transmission of radiant energy being determinedby the dimension of'the displacement plate along the axis of the beam ofradiant energy used, and being reproducible because of the constantdimensions of the displacement plate, and being substantiallyindependent of the shape and optical condition of the surface of thecontainer of the fluid of unknown concentration, said change intransmission of radiant energy making possible calculation of theunknown concentration of energy-absorbing molecules being measured.

References Cited in the file of this patent UNITED STATES PATENTS1,635,470 Exton July 12, 1927 1,877,501 EXton Sept. 13, 1932 1,954,925Exton Apr. 17, 1934 2,073,223 Rose Mar. 9, 1937 2,645,971 Herbst July21, 1953 2,844,066 Friel July 22, 1958

